Figure captions
Figure 1. Schematic illustration depicting cytosolic protein
delivery using ultrasound-guided vaporization of phase-change
nano-droplets. (A) The chemical structure of the protein anchoring
reagent that anchors the protein onto the nano-droplets (DPPE-PEG-PyDTP)
and the corresponding anchoring reaction. (B) Chemical structures of the
nano-droplet modifiers for antibody modification (DPPE-PEG-Biotin),
stabilization (DPPE-PEG-OMe), and labeling (DPPE-PEG-TAMRA). (C)
Schematic of cellular uptake and ultrasound-guided intracellular
vaporization of the droplets carrying cargo proteins for cytosolic
delivery through endosomal escape.
Figure 2. Characterization of droplets for cytoplasmic protein
delivery. (A) The concentration of the released β-galactosidase from the
droplets after incubation with various concentrations of reduced
glutathione (GSH). (B) Dynamic light scattering intensity of the
droplets after down-sizing using an extruder. (C) Confocal microscopic
images of DLD1 cells treated with nano-droplets modified with
anti-epiregulin antibody and (D) without anti-epiregulin antibody. The
antibody was labeled with a fluorescent dye (Alexa Fluor 647: AF647)
(pink). The nano-droplets were labeled with DPPE-PEG-TAMRA (orange).
Scale bar: 10 μm.
Figure 3. Fluorescence confocal microscopic images of cells
after ultrasound-induced cytosolic delivery of β-galactosidase (β-Gal).
(A) DLD1 cells treated with phase-change nano-droplets (PCNDs) carrying
β-Gal and ultrasound, (B) those treated with β-Gal-carrying PCNDs
without ultrasound, and (C) those treated with PCNDs not carrying β-Gal
and ultrasound were observed after staining with a fluorescent substrate
for β-Gal (TokyoGreen-βGal) and a endosomal and lysosomal staining dye
(Lysotracker). (D) Intact cells were also observed as the control in the
same way. The fluorescence images for TokyoGreen-βGal (green) and
Lysotracker (cyan) were merged along with the differential interference
contrast images. Scale bar: 25 μm.
Figure 4. Viabilities of cells after ultrasound (US)-induced
cytosolic delivery of saporin (Sap). DLD1 cells were treated with the
nano-droplets (PCNDs) with or without Sap and then were exposed to
ultrasound (US) or not. In the same way, the viability of intact cells
was examined. The viability was normalized to that of the intact cells.
Each bar represents the mean ± S.E. (n = 3). *p < 0.01 and **p
< 0.05 versus the cells treated with both PCNDs carrying Sap
and US (t-test).